Biotinylation and (Theoretical) Immunoprecipitation of Cyclohexanepentanoic Acid

Day 1

1. Inoculate Cultures in 50 mL Tubes (whatever cultures you are interested in probing) with and without the compound you are sensing.

2. At the same time set up a biotinylation reaction as follows, dissolving all solutions in 80% DMSO:

Add10 uL of pure cyclohexanepentanoic acid to 167.10 uL of 80% DMSO
Make up a solution 500 mM EDAC in 80% DMSO. (Dissolve 10 mg of EDAC in 0.1 mL of 80% DMSO) Add 12.5 uL of the EDAC to the cyclohexanepentanoic acid solution you made. Don’t keep the EDAC out very long it is very hydroscopic and must be kept away from moisture. Store the solution you make at -20C in the biotin box!
Make up a stock solution of 100 mM Biotin by weighing it out into a 1.5 mL Epindorf on an analytical balance (be careful when you do this) spin down the tube then add 80% DMSO as before. Again store this solution at -20C in a tightly sealed container.
pH the solution using 5% HCl (found in the acid cabinet) to pH 4-7. Optimal pH of the solution appears to be between 5-5.5. Add 2 uL of the acid to the solution and mix well. Take a few microlitres of the solution and spot it onto a piece of pH paper. Repeat if necessary
Let the solution sit at room temperature O/N.

3. Set up a second reaction for control with biotin only in 80% DMSO.

Add 12.5 uL of the Biotin solution that you made into 187.5 uL of 80% DMSO. pH the solution to roughly the same pH as your reaction using 5% HCl (using ~2uL) and let it sit O/N like the other reaction at room temperature.

FINAL CONCENTRATION OF THE SOLUTION

10 mg of cyclohexane pentanoic acid (or 54mM) 5 mM EDAC 5 mM Biotin

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